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SARSeq, a robust and highly multiplexed NGS assay for parallel detection of SRAS-CoV2 and other respiratory infections

ABSTRACT: During a pandemic, mitigation as well as protection of system-critical or vulnerable institutions requires massively parallel, yet cost-effective testing to monitor the spread of agents such as the current SARS-CoV2 virus. Here we present SARSeq, saliva analysis by RNA sequencing, as an approach to monitor presence of SARS-CoV2 and other respiratory viruses performed on tens of thousands of samples in parallel. SARSeq is based on next generation sequencing of multiple amplicons generated in parallel in a multiplexed RT-PCR reaction. It relies on a two-dimensional unique dual indexing strategy using four indices in total, for unambiguous and scalable assignment of reads to individual samples. We calibrated this method using dilutions of synthetic RNA and virions to show sensitivity down to a few molecules, and applied it to hundreds of patient samples validating robust performance across various sample types. Double blinded benchmarking to gold-standard quantitative RT-PCR performed in a clinical setting and a human diagnostics laboratory showed robust performance up to a Ct of 36. The false positive rate, likely due to cross contamination during sample pipetting, was estimated at 0.04-0.1%. In addition to SARS-CoV2, SARSeq detects Influenza A and B viruses as well as human rhinovirus and can be easily expanded to include detection of other pathogens. In sum, SARSeq is an ideal platform for differential diagnostic of respiratory diseases at a scale, as is required during a pandemic.

ORGANISM(S): Homo sapiens

PROVIDER: GSE163688 | GEO | 2020/12/23

REPOSITORIES: GEO

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Project description:There is an urgent need for robust and high-throughput methods for SARS-CoV-2 detection in suspected pa-tient samples to facilitate disease management, surveillance, and control. Although nucleic acid detection methods such as RT-PCR are the gold standard, during the current pandemic the deployment of RT-PCR tests has been extremely slow, and key reagents such as PCR primers, and RNA extraction kits are at critical shortages. Rapid point-of-care viral antigen detec-tion methods have been previously employed for the diagnosis of respiratory viruses such as influenza and respiratory syn-cytial viruses. Therefore, the direct detection of SARS-CoV-2 viral antigens in patient samples could also be used for diagno-sis of active infection and alternative methodologies for specific and sensitive viral protein detection should be explored. Targeted mass spectrometry techniques have enabled the identification and quantitation of a defined subset of pro-teins/peptides at single amino acid resolution with attomole level sensitivity and high reproducibility. Herein we report a tar-geted mass spectrometry assay for the detection of SARS- CoV-2 spike protein and nucleoprotein in a relevant biologi-cal matrix. Recombinant full-length spike protein and nucleoprotein were digested and prototypic peptides were selected for parallel reaction monitoring (PRM) quantitation using a high resolution Orbitrap instrument. A spectral library, which con-tained 7 proteotypic peptides (4 from spike protein and 3 from nucleoprotein) and the top 3 to 4 transitionsMS2 spectra, was generated and evaluated. From the original spectral library, we selected 2 best performing peptides for the final PRM assay. The assay was evaluated using mock test samples containing inactivated SARS-CoV-2 virions, added to in-vitro de-rived mucus. The PRM assay provided a limit of detection (LOD) of ~200 attomoles and a limit of quantitation (LOQ) of ~ 390 attomoles. Extrapolating from the test samples, the projected titer of virus particles necessary for detection of SARS-CoV-2 spike and nucleoprotein detection was approximately 2E5 viral particles/mL, making it an attractive alternative to RT-PCR assays. Potentially mass spectrometry-based methods for viral antigen detection may deliver higher throughput and could serve as a complementary diagnostic tool to RT-PCR. Furthermore, this assay could be used to evaluate the pres-ence of SARS-CoV-2 in archived or recently collected biological fluids, in-vitro derived research materials, and wastewater samples

Project description:The ongoing pandemic of coronavirus disease 2019 (COVID-19), which results from the rapid spread of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is a significant global public health threat, with molecular mechanisms underlying its pathogenesis largely unknown. Small non-coding RNAs (sncRNAs) are known to play important roles in almost all biological processes. In the context of viral infections, sncRNAs have been shown to regulate the host responses, viral replication, and host-virus interaction. Compared with other subfamilies of sncRNAs, including microRNAs (miRNAs) and Piwi-interacting RNAs (piRNAs), tRNA-derived RNA fragments (tRFs) are relatively new and emerge as a significant regulator of host-virus interactions. Using T4 PNK‐RNA‐seq, a modified next‐generation sequencing (NGS), we recently found that nasopharyngeal swabs (NPS) samples from SARS-CoV-2 positive and negative subjects show a significant difference in sncRNA profiles. There are about 166 SARS-CoV- 2-impacted sncRNAs. Among them, tRFs are the most significantly affected and almost all impacted tRFs are derived from the 5’-end of tRNAs (tRF5). Using a modified qRT-PCR, which was recently developed to specifically quantify tRF5s by isolating the tRF signals from its corresponding parent tRNA signals, we validated that tRF5s derived from tRNA GluCTC (tRF5-GluCTC), LysCTT (tRF5-LysCTT), ValCAC (tRF5-ValCAC), CysGCA (tRF5-CysGCA) and GlnCTG (tRF5-GlnCTG) are enhanced in NPS samples of SARS-CoV2 patients and SARS-CoV2-infected airway epithelial cells. In addition to host-derived ncRNAs, we also identified several sncRNAs derived from the virus (svRNAs), among which a svRNA derived from CoV2 genomic site 346 to 382 (sv-CoV2-346) has the highest expression. The induction of both tRFs and sv-CoV2-346 has not been reported previously, as the lack of the 3’-OH ends of these sncRNAs prevents them to be detected by routine NGS. In summary, our studies demonstrated the involvement of tRFs in COVID-19 and revealed new CoV2 svRNAs.

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SARSeq, a robust and highly multiplexed NGS assay for parallel detection of SRAS-CoV2 and other respiratory infections

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